Peptides and proteins are biological molecules existing normally in organisms. The elucidation of physiological activities and of mechanisms these biological molecules are of much interest to the fields of biochemistry, physiology and medicine. The synthesis of peptides or proteins having specific amino acid sequences has increased due to the use of automated peptide synthesizers. Studies In the above mentioned fields are expected to show sharp progress, if peptides or proteins having specific amino acid sequences can be synthesized with higher purities. However, present peptide synthesis methods produce a relatively large number of impurities, as well as target compound. Therefore all important objective of a solid-phase peptide synthesis method is to recover the target peptide alone from impurities with high speed and high yield.
Gel flirtation, high-performance liquid chromatography, and combination thereof are presently for the purification of peptides or proteins synthesized by a solid-phase method (R. B. Merrifield, J. Am. Chem. Soc., 85, 2149 (1963)). For some special peptides and proteins, affinity chromatography may be an effective purification method, but not a perfect one. The reason is that some of the amino acid deleted peptides may have an affinity (even a low degree) for the supports used in affinity chromatography, the amino acid deleted peptides being synthesized as impurities during the solid-phase synthesis as part of the resultant peptide mixture.
The peptides are synthesized by a step-wise elongation. For example, in case of condensation of a 50 residue peptide with the condensation reaction yield of 99%, the theoretical synthetic yield reaches to 60%. Condensation reaction yield over 99% can not always be obtained since the condensation reaction depends on the sequence of peptides. As a result, amino acid deleted peptides pile up as impurities by incomplete condensation reactions.
A capping by acetic anhydride is performed after every condensation reaction to terminate further elongation of peptide chains of a non-target sequence and to avoid further production of amino acid deleted peptides. After the coupling of the final amino acid, only the peptide having a target amino acid sequence will have an amino group at its N-terminus.
Several reports on purification methods using the N-terminus amino group have been published. (See, for example, R. Camble, R. Garner and G. T. Young, Nature (London), 217, 247 (1968); K. Suzuki, Y. Sasaki and N. Endo, Chem. Pharm. Bull., 24, 1 (1976); D. S. Kemp and D. G. Roberts, Tetrahedoron Lett., 4269 (1975); T. Weiland, C. Birr and H. Wissenbach, Angew. Chem., Int. Ed., Engl., 8, 764 (1969), H. Wissman and R. Geiger, Angew. Chem., Int. Ed., Engl., 9, 908 (1970); R. B. Merrifield and A. E. Bach, J. Org. Chem. 43, 4808 (1975); T. J. Lobl, R. M. Deibel and A. W. Yen, Anal. Biochem., 170, 502 (1988); H. Ball, C. Grecian, S. B. H. Kent and P. Mascagni, in "Peptides", J. E. Rivier and G. R. Marshall, Eds., ESCOM, Leiden 1990 pp 435). However, none of these methods has been able to achieve effective one-step separation, instead complicated separation processes are required.
Another method has been developed in which the target peptide alone is absorbed to a phenyl-mercury column by attaching cysteine-methionine to the N-terminus of the synthesized peptide, and using the SH group of the cysteine. Subsequent to the separation, the methionine-peptide bond is cleaved by BrCN to yield the target peptide. (D. E. Krieger, B. W. Erikson and R. B. Merrifield, Proc. Natl. Acad. Sci. U.S.A., 73, 3160 (1976)) However, this method has a limitation of being not applicable to peptides containing methionine.